U.S. Pat. No. 4,946,953 to Okuma and U.S. Pat. No. 4,055,510 to Peska et al. provide an understanding of the background of the invention. Both teach the making of porous, swellable, spherical, reconstituted cellulose gel particles. U.S. application Ser. No. 07/374,281, filed Jun. 30, 1989 by two of us, Scarpa and Beavins, issued as U.S. Pat. No. 5,245,024, Sep. 14, 1993, directly relates to the problems addressed by this application. The substance of that application was published Jan. 10, 1991 with the entry into the National Phase of the corresponding PCT Application No. PCT/US90/03716 Publication No. W091/00297 is incorporated by reference herein.
Cellulose and cellulose derivatives long have been used as chromatographic supports and as filtration media. General chromatographic uses include analytical and preparative column liquid chromatography, thin-layer chromatography, ion exchange and gel chromatography, and chelation and affinity sorbents. Additionally, cellulose particles have numerous other uses in the pharmaceutical, food, and cosmetics industries.
Cellulose is a naturally-occurring polymer of 1,4-beta-linked glucose monomers. In the native state, polymeric glucose chains are extensively hydrogen-bonded to each other in some regions and less hydrogen-bonded in others. The regions of relatively high hydrogen bonding are generally referred to as "microcrystalline regions", while the less hydrogen bonded regions are referred to as amorphous regions. The newly discovered smooth skin is neither wholly crystalline nor purely amorphous, but, under illumination with polarized light, reveals some orientation, indicating a degree of order in the skin, which we speculate is related to the formation at the interface of solvent and viscose, and related to the property of the skin to change its chemical activity.
Originally cellulose fibers were used in chromatography, but the need to improve the rate of flow led to the rise of microcrystalline cellulose as the medium of choice. Procedures typically used to prepare microcrystalline cellulose generally result in aggregated particles which may be comminuted by grinding and then separation by particle-size, commonly referred to as "classification", yields a medium suitable for some chromatographic purposes. The resulting individual microcrystalline cellulose particles are relatively irregularly shaped and fragile, which features adversely affect the use of these types of materials in chromatographic beds or columns. Because microcrystalline cellulose tends to break down and generate fines, resulting sorbent beds are prone to clogging and compacting when columns are subjected to elevated pressure. These drawbacks can result in unacceptable flow characteristics and poor chromatographic separations.
In order to overcome swelling and poor flow characteristics, Okuma reports in U.S. Pat. No. 4,946,953 the use of crosslinked cellulose beads as a desalting chromatographic medium for protein purification. Those beads were resistant to medium pressure and offered a reasonably good flow rate.
The Scarpa-Beavins procedure as disclosed in U.S. Pat. No. 5,245,024 produces spherical, dense, uncrosslinked beads of a narrow size distribution which resist moderate pressures when tested with beads of 30 to 90 microns diameter. These beads typically do, however reveal some surface porosity in the form of cracks, voids, and unevenly distributed holes or channels of varying diameters. Such structural irregularities can lead to loss of resolution when separating biomolecules or other macromolecules due to their diffusion into the interior of the beads.
As discussed earlier, crosslinking yields a new polymer, consisting of crosslinked cellulose chains, thereby rendering the beads more mechanically stable, but also such crosslinking increases the cost of the support, complicating the manufacturing processes, thereby limiting the general utility of use of such support.
Porous, uncrosslinked cellulose particles, such as disclosed in Peska, when placed in aqueous solutions typically swell significantly. Swollen, porous cellulose beads are sensitive to changing ionic strengths in elating buffers and solvents and do not withstand high pressure gradients. As a result, known swellable cellulose supports may therefore be used only within a specified range of ionic strengths. If this specific range of ionic strengths is exceeded, the swelled cellulose particles compact or shrink which results in very poor flow characteristics and leads to either poor chromatographic separations or to no separations at all.
A great many methods of preparing spherical cellulose particles are known. Japan patents 73'21m738 and 73'60,753 extrude a viscose at high speed through a nozzle into a spinning acidic coagulation bath. The thermal decomposition of the sodium cellulose xanthate results in porous particles as disclosed in Peska et al, U.S. Pat. No. 4,055,510. Scarpa-Beavins U.S. Pat. No. 5,245,024 application Ser. No. 07/374,281 discloses a process for the making of substantially spherical, high-density-cellulose particles by the steps of forming a stable emulsion of high-molecular-weight viscose, using at least one emulsifying agent and a water immiscible liquid carrier of suitable viscosity at a temperature typically between 20 and 30 degrees C., by slowly aging and coagulating the cellulose xanthate while the beads are kept in suspension by stirring. Finally, cellulose is regenerated by contacting the coagulated beads with acid solutions. Beads made according to the method just described, being solid and microporous with relatively few larger voids and holes, do not shell appreciably when contacting aqueous solutions of varying salt concentration.
Never-the-less when following the teachings of the Scarpa-Beavins U.S. Pat. No. 5,245,024 to produce larger batches (represented by the Examples 1), that is, 50 liter batches and larger, it was found that the time needed for coagulation of beads becomes prohibitively long. In addition, it is evident from electron micrographs, such as FIG. 5b of a fractured bead, that the interior of the beads is sparsely populated with holes, some of which reach 5,000 .ANG. (Angstroms) in size, and channels connecting holes with the surface. In certain instances, as where ligands are attached, chromatographic separation can be optimized when substrate/sorbent interactions take place exclusively on the outside surface of the bead. In such cases, the presence of any holes of a size that may accommodate a substrate molecule cannot be tolerated; otherwise diffusion-based interferences may adversely affect resolution of pure compounds. The inventions for which this continuing application is submitted arose from our efforts to eliminate the holes and voids in the final bead product, that may have access to the liquid phase as well as to shorten the reaction time required for coagulation of the beads. Since the filing for Scarpa-Beavins U.S. Pat. No. 5,245,024, we have learned that a modification of the methods disclosed therein results in a new form of cellulose bead, spheroidal in shape, with a smooth, strong skin, which skin, of oriented cellulose, is impervious to nitrogen (under conditions of the BET method for measuring surface area), and effectively non-swelling in aqueous solutions.